Abstract: A shortcoming in microbiome research is the lack of reference standards
to control biases introduced by differential DNA extractability,
interference with amplification, library preparation, next-generation
sequencing platforms, and data analysis. The aim of this research is to develop and test novel molecular barcodes for use as spike-in
reference standards in the form of oligonucleotides or recombinants in
microbiome studies.

Evaluation of 16S rRNA and Shotgun Metagenomic Analytical Methods for Community Profiling using ATCC® Microbiome Standards

Abstract: Complexities involved in 16S rRNA and shotgun metagenomic analysis methods pose significant challenges in microbiome research as various biases can be introduced during PCR amplification, library preparation, sequencing, and analysis. One of the primary obstacles in assay standardization is the limited availability of reference materials. To support this need, we developed microbiome reference standards from fully sequenced and characterized ATCC® strains and evaluated their use in a proof-of-concept study. Here, four standards were created comprising mixtures of 10 or 20 genomic DNAs in equal or staggered quantities prepared from a diverse set of bacteria that were selected based on relevant attributes such as Gram stain, genome size, GC content, and other special characteristics. Initially, we performed an inter-laboratory comparison of the 16S rRNA V4 region from three different commercial laboratories. Analysis of the resulting sequencing data using One Codex revealed variability in the number of true positives and false positives as well as the relative abundances. A subsequent comparison of three different regions of the 16S rRNA gene—V1V2 (27f-YM+3 338R), V3V4 (341F & 806R), and V4 (515F & 806R)—revealed that only the analysis of the V1/V2 region using One Codex was able to profile the bacteria to the species level. Following this analysis, we evaluated a shotgun metagenomics approach and compared it to the 16S rRNA V1/V2 results. Here, the shotgun metagenomics approach showed a high correlation between the expected vs. observed ratios as compared to the 16S rRNA results, which had variable correlation for three standards with equal and staggered ratios. These results demonstrate that 16S rRNA community profiling of the V1V2 region and the shotgun metagenomic approach could identify bacterial strains to the species level, with the latter method generating relative abundances statistically close to the expected. Taken together, this proof-of-concept study demonstrates the potential use of ATCC® Microbiome Standards in the identification of potential biases and methodology drawbacks associated with microbiome studies.

Development and Evaluation of Mock Microbial Community Standards based on DNA Extractability from Bacteria

Abstract: The complexities involved in 16S rRNA-based and metagenomics analysis methods pose significant challenges for standardization as bias can be introduced during DNA extraction, amplification, library preparation, sequencing, and bioinformatics analysis. One of the primary challenges in assay standardization is the limited availability of reference materials. To address this issue, we evaluated the use of two mock microbial communities in the form of lyophilized, whole-cell standards as full-process controls.

Development of an Assembly and Delivery Platform for DNA Circuit Construction in Saccharomyces cerevisiae

Abstract: Complex behavior within eukaryotic cells manifest from layered regulatory networks changing the transcription of many genes. To systematically study these pathways by modulating individual components—or in the case of synthetic biology, building new network architectures by creating DNA circuit—it is critical to control multiple genes simultaneously under tightly regulated or inducible expression. In the case of network construction in Saccharomyces cerevisiae, there has been a lack of both suitable, well-characterized parts (promoters and regulators) as well as a standardized platform for DNA assembly and delivery of gene circuits. Here, we present a framework for building gene circuits as well as a set of fully characterized DNA parts for use in Saccharomyces cerevisiae. The entire procedure of building a gene circuit from more than 10 basic parts took less than 5 days with only a workload of 1-3 hours per day. A diverse promoter collection comprising five different types was generated: constitutive, yeast native inducible, synthetic inducible, synthetic promoters regulated by activators, and synthetic promoters regulated by repressors. Altogether, the range of promoters span 2-fold to 105-fold expression above the background, the new inducible systems allow 11-fold change in expression, and the activators/repressors show a maximum 35-fold and 45-fold change of expression. This study demonstrates the feasibility for the quick and easy construction of gene circuits for delivery into S. cerevisiae and the utility of a fully characterized set of diverse promoters, activators, and repressors. This assembly system combined with DNA parts will be useful for constructing large-scale gene circuit libraries with reliable gene expression and for designing logic operations for a complex network in S. cerevisiae. Moreover, we anticipate that our system will allow for the controlled study of multi-step pathways by enabling manipulation of single protein expression.

Development and Characterization of an In Vitro Co-culture Angiogenesis Assay System (Angio-Ready™; ATCC® ACS-2001-2™) Using hTERT-immortalized Cells for High-throughput Drug Screening

Abstract: Angiogenesis is a multi-step physiological process that is involved in a large number of normal and disease state processes; in vitro angiogenesis models provide very useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays are significantly more heterogeneous and more closely resemble capillaries than tubules formed in Matrigel®1. Current co-culture models using primary cells have donor variability, as well as inconsistent results due to lot-to-lot variation. In this study, we established an in vitro co-culture model system consisting of an assay-ready mixture of an hTERT-immortalized human aortic endothelial cell line (TeloHAEC-GFP) and an hTERT-immortalized adipose-derived mesenchymal stem cell line (hTERT-MSC) in a specially formulated medium, the Angio-Ready™ angiogenesis medium with vascular endothelial growth factor (VEGF) supplement. Both cell lines were immortalized by a single agent, hTERT (human telomerase reverse transcriptase), and have been well-characterized to verify that the cells retain the most important characteristic of their parental counterparts. The new co-culture system forms functional tubular structures in less than 7 days. Further, the hTERTMSC cells that surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining (a marker of smooth muscle cells), indicating that the system has physiological relevance. Next, we tested the new assay with compounds that impact angiogenesis; the results demonstrated that the angiogenesis system responds positively to elevated doses of VEGF and negatively to increasing concentrations of suramin, a known VEGF inhibitor. More importantly, the tubular formation efficiency is reduced or blocked by well-known cancer drugs such as sunitinib (Sutent®) and bevacizumab (Avastin®), both of which target the VEGF pathway. Notably, results show the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots of the Angio-Ready™ system with ramucirumab (Cyramza®), which also targets the VEGF pathway. Finally, the Angio-Ready™ system was further validated in a high-throughput format by using 4 HIF-1(hypoxia inducible factors-1) inhibitors, which have antiangiogenic properties identified by high-throughput screening methods; data showed the results of the Angio-Ready™ system is well matched with other screening methods. What's more, the Angio-Ready™ system can be assayed in as few as 3 days. Therefore, the co-culture models developed using the hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying cardiovascular biology, drug screening and tissue engineering.

New type of drug-resistant isogenic cell model created by CRISPR genome editing

Abstract: A mutant BRAF gene can lead to uncontrolled cell growth through overactivation of the RAS-RAF-MAPK signaling pathway. The BRAFV600E mutation occurs in approximately 40% to 50% of melanomas. Although current BRAF inhibitors have been used to successfully treat melanomas containing the BRAFV600E mutation, patients often become resistant to BRAF inhibitors within a few months. A number of clinical studies have indicated that secondary mutations in RAS or NF1 are associated with BRAF resistance. However, due to the genetic heterogeneity commonly observed in tumors, it is unclear if those secondary mutations already existed within low percentage subclones, or if they were acquired through drug treatment. Further, it has yet to be determined whether such genetic variants are only associated with resistance, or whether they actually cause the BRAF inhibitor resistance. In this study, we used genome editing CRISPR technology to generate two drug-resistant melanoma cell lines (ATCC® CRL-1619IG-2™ and CRL-1619IG-1™) that contain NRASQ61K or KRASG13D mutations. These isogenic lines were derived from the parental A375 (ATCC® CRL-1619™) melanoma cell line, which naturally contains BRAFV600E. When compared to the parental line, the isogenic cell models demonstrated that genetically modified NRAS or KRAS genes at the endogenous level directly leads to significant resistance to BRAF inhibitors.

Single guide RNAs (sgRNAs) were designed and built to guide Cas9 to bind and cut desired regions in the NRAS or KRAS gene targets. The parental cell line A375 was co-transfected with the single guide and CRISPR all-in-one plasmid alongside a donor plasmid. Transfected cells were sorted into single cells and expanded for subsequent screening of desired gene mutation events. The introduction of the NRASQ61K or KRASG13D mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic and transcriptional levels. Drug responses to BRAF-specific inhibitors and non-specific chemotherapy drugs were compared between RAS isogenic A375 cell lines and parental A375 cell line in 2D and 3D culture environments. Testing results demonstrated that the isogenic cell lines created by CRISPR showed significant resistance to BRAF inhibitors in comparison to the parental control in both 2D and 3D culture environments. These two novel in vitro cell models with endogenous level RAS mutants provide direct biofunctional evidence that acquiring a drug-resistant gene drives tumor cell survival under targeted therapeutic treatment.

Abstract: Tumor development begins with mutational changes to the genetic makeup of a cell; tumor progression is not solely determined by the mutated cell, but also by the tumor's microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been determined to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblasts (CAFs) differ from normal-associated prostate fibroblasts (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we characterized three prostate-derived cells: CAFs, NAFs, and prostate epithelial cells (PrEs); all three lines were immortalized by(human telomerase reverse transcriptase (hTERT) alone, and have been continuously passaged for more than 40 PDL in our hands. Our data shows that the hTERT-immortalized CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (α-SMA), while neither cell line expresses epithelial markers such as CK14. Both CAFs and NAFs also express elevated levels of α-SMA upon TGF-β stimulation. All three prostate-derived cells weakly express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumor cell growth better than NAF conditioned media. In conclusion, CAFs, NAFs, and immortalized PrEs may provide a very valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies.

Abstract:In vitro cell culture models are becoming increasingly complex in an effort to better mimic in vivo physiology and enhance their predictive power and relevance in areas including absorption, distribution, metabolism, excretion, and toxicity assays (ADMET), drug discovery, phenotypic screening, developmental biology, and basic research. One major advance in model development has been the recognition of the critical role of the cell microenvironment including the presence of extracellular biomolecules, cell-to-cell interactions, and physical properties of the substrate. These elements can influence a variety of cellular functions including cellular metabolism, differentiation potential, gene/protein expression, drug susceptibility, proliferation, and survival, typically with extracellular matrix (ECM)- and cell type-specificity. Herein we report the development of a “universal” CellMatrix Basement Membrane ECM that provides a suitable microenvironment to support a wide variety of cell biology applications. Our results show that with optimized protocols the ECM permitted the long-term 3D culture of primary- and iPSC (induced pluripotent stem cells)- derived gastric and intestinal (GI) organoids from human and mouse cells and significantly enhanced spheroid forming efficiency in multiple continuous cancer lines. ATCC CellMatrix Basement Membrane also supported angiogenic vessel formation in engineered, immortalized and primary human endothelial cells under co-culture conditions; increased cytochrome P450 enzyme activity and multidrug resistance-associated protein 2 (MRP2) expression in “sandwich” culture of primary human hepatocytes; and allowed the routine culture of iPSCs and NPCs (neural progenitor cells) under serum- and feeder-free conditions while maintaining and promoting the capacity for terminal differentiation into neurons and beating cardiomyocytes. These results demonstrate that ATCC CellMatrix Basement Membrane provides a suitable biological matrix for advanced, physiologically relevant in vitro cell- based models and assays.

Abstract: Growing concern over bacterial food contamination has led to increased examination of food testing protocols in today’s industry. Currently, the use of bacterial strains as positive controls in testing protocols is not widely practiced for fear of cross-contaminating samples. Due to ongoing scrutiny of food testing methodology and growing regulations under the Food and Drug Administration (FDA) Food Safety Modernization Act, it is imperative to have control strains with unique, easily detectable traits that distinguish positive control strains from actual food contaminants, diminishing the fear of cross-contamination and improving current practices. In this study, we developed GFP reporter-labeled Shiga toxin-producing Escherichia coli for use as controls in QC testing.

Abstract: Herpes simplex virus (HSV-1 and HSV-2) causes a wide range of clinical manifestations that result in lifelong infections. Quantitative PCR (qPCR) assays are routinely used for the detection of HSV-1 and HSV-2 infections in clinical samples. However, the accuracy of a qPCR assay is dependent upon the generation of a standard curve using a positive control with a known genome copy number. Moreover, an independent positive control is required to monitor variations in assay performance for molecular assays. ATCC has developed HSV-1 and HSV-2 quantitative molecular standards for use as controls for the detection and quantification of these viruses from clinical samples.

Development of Synthetic Molecular Standards for Hepatitis B and Hepatitis C virus

Abstract: Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) is a major health concern and affects millions of people worldwide. Patients are routinely monitored by quantitative RT-PCR (qRT-PCR) for the presence of HCV RNA or by qPCR for HBV DNA in blood. Since these viruses are difficult to culture in vitro, obtaining control material for these molecular-based assays is a challenge. To address this problem, ATCC has developed HBV and HCV specific quantitative synthetic molecular standards for use as controls for the detection and quantification of these viruses from clinical samples.

Abstract:in vivo studies have shown that kidney membrane transporters play a key part in drug disposition and renal clearance. One such transporter is OAT1 (SLC22A6), which is critical for maintaining homeostasis of endogenous substances. This makes OAT1 a good transporter to assay for drug interactions with the kidney. Unfortunately, primary cells lose OAT1 expression in culture, and transiently expressed OAT1 has great variations between production lots, which make data hard to interpret. In our study we have generated HEK 293T/17 cells that stably overexpress the OAT1 gene driven by the human elongation factor-1 alpha (EF1α) promoter. After confirming the mRNA expression by RT-PCR, we performed immunostaining that indicated OAT1 is correctly trafficked to the membrane. Most importantly, we validated that the overexpressed OAT1 transporter has normal transport activities by using 5-carboxyfluorescein (5-CF) and para-aminohipurate (PAH; data not shown) uptake assays, and that the uptake can be inhibited by the well-known inhibitors probenecid and novobiocin. Both inhibitors responded in a dose dependent manner for 5-CF uptake with IC50 values between 5-16 μM. Even at higher passages, the cell line retained the functionality of OAT1. Overall, our data has shown that this modified cell line is a very useful in vitro tool for testing regulation of OAT1 membrane transporter activity in kidney cells.

The Generation of an EML4-ALK Fusion NSCLC Isogenic Cell Line Relevant for Drug Discovery and Development

Abstract:Recent studies show that tumor cells derived from a subset of patients with non-small-cell lung cancer (NSCLC) harbor the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene; the result of a Paracentric chromosomal inversion on the short arm of chromosome 2. The EML4-ALK oncogene, like other ALK fusion oncogenes, is a druggable target that is responsive to ALK inhibitors. However, there is a lack of EML4-ALK in vitro models for drug screening. Here we set out to generate an isogenic EML4-ALK fusion non-small cell lung cancer model in the A549 lung cancer cell line, which harbors other naturally occurring genomic aberrations inherent in non-small cell lung cancer. This model could serve as a clinically relevant drug screening cell model. In this study, we utilized the CRISPR/Cas9 genome editing platform to target endogenous loci in human cells and create the intended genomic translocation event. By employing sgRNAs-Cas9 constructs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant genomic rearrangements that resulted in the expression of EML4–ALK fusions. Breakpoint junction analysis tested after sgRNA-CRISPR/Cas9 mediated genomic DNA cleavage in A549 cells showed the successful creation of the EML4-ALK fusion found in tumor cells from a subpopulation of NSCLC patients. Furthermore, single clonal isolation and functional screening demonstrated that the EML4-ALK isogenic cell line was sensitive to ALK inhibitors relative to the parental A549 cell line. This newly developed EML4-ALK isogenic lung cancer cell line could provide a very useful tool for oncology drug discovery and development.

Development and Characterization of Mesenchymal Stem Cells Immortalized by hTERT and their Use in Co-culture Models for Wound Healing and Angiogenesis

Abstract:This study will suggest that mesenchymal stem cells (MSC) with their inherent multi-potency are a better alternative to fibroblasts in certain co-culturing situations. Cell-based assays that use primary cells do offer better predictability, but they are hampered by the finite life span of primary cells, which causes inconsistency in the results from donor variation. hTERT-immortalized cell lines solve this problem by offering the functionality of a primary cell with the longevity of a cell line. In this study we characterize the functionality of an hTERT immortalized adipose tissue-derived MSC cell line (hTERT-MSC) and illustrate its use in two co-culturing applications: wound healing and angiogenesis We confirmed that primary keratinocytes and an hTERT-immortalized keratinocyte cell line, Ker-CT, are able to fully differentiate into skin equivalents in an air-liquid interface (ALI) 3D culture model, when co-cultured with hTERT-MSCs. To confirm the functionality of the co-culture models, both primary keratinocytes and Ker-CT ALI co-cultures were subjected to a scratch assay. Re-epithelialization occurred in both cell lines. Additionally, we established an in vitro angiogenesis co-culture model system using TeloHAEC-GFP (an immortalized aortic endothelial reporter cell line stably expressing GFP), and hTERT-MSCs. Results show that the new model can form tubular structures in less than 7 days instead of 14 days compared to co-culture with fibroblasts, and also responds effectively to VEGF stimulation and drug treatments. Furthermore, immunofluorescence staining shows that cells surrounding the tubular structures stain positive for αSMA supporting the physiological relevance of this in vitro model system. The co-culture models developed by using hTERT-MSCs in this report provide a more consistent and robust in vitro co-culture system for studying wound healing and vascular biology for drug screening and tissue engineering

Development of Defined Conditions for Expansion and Dopaminergic Neuron Differentiation of iPSC-derived Neural Progenitor cells

Abstract: Human induced pluripotent stem cells (iPSCs) have the capacity to differentiate into all of the somatic cells types and hold great promise for both regenerative medicine and drug discovery. A need for better tools to address neurological disease modeling and neuro-toxicology screening exists. We have developed a scalable process that allows for the generation of large quantities of neural progenitor cells (NPCs) derived from normal and Parkinson’s disease iPSC lines, along with a serum-free defined NPC expansion medium and dopaminergic differentiation medium.
To validate the process of NPC derivation, we generated NPCs derived from iPSCs that were reprogrammed with the Sendai virus from the following sources: human foreskin fibroblasts (HFF-1), human CD34+ cells, and human fibroblasts from a patient with Parkinson’s disease. Compared to Parkinson’s disease patient-derived NPCs, both normal NPC lines demonstrated greater proliferative capacity. Moreover, our CD34+ cell-derived NPCs possessed better tri-lineage differentiation efficiency than that of fibroblast-derived NPCs although all three types of NPCs were capable of differentiating into dopaminergic neurons, astrocytes, and oligodendrocytes.

Development of Improved Synthetic Molecular Standards for Norovirus Genogroups I and II

Abstract: Noroviruses (NoV) are the most common cause of epidemic gastroenteritis, accounting for 95% of viral gastroenteritis outbreaks worldwide. NoV detection is difficult because they are genetically heterogeneous and cannot be grown in cell culture. The principle detection method utilized by diagnostic laboratories is quantitative RT-PCR (qRT-PCR). The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known genome copy number. To that end, we have developed quantitative synthetic standards that include conserved sequences from NoV GI and NoV GII for the detection and quantification of NoV from either clinical, food, or environmental samples. These quantitative synthetic molecular standards provide well-characterized reference materials for the detection and quantification of NoV by qRT-PCR. Further, they exhibit excellent compatibility with numerous published NoV assays and can be used as controls for assay development, verification, and validation.

Abstract: In this study, we compared primary keratinocytes (ATCC® No. PCS-200-010) to hTERT immortalized keratinocytes (Ker-CT; ATCC® No. CRL-4048™), co-cultured with either primary fibroblasts (ATCC® No. PCS-201-010), primary adipose-derived mesenchymal stem cells (MSCs; ATCC® No. PCS-500-011), hTERT-immortalized fibroblasts (BJ-5ta; ATCC® No. CRL-4001™), or hTERT-immortalized MSCs (hTERT-MSCs; ATCC® No. SCRC-4000™). We confirmed that both primary keratinocytes and Ker-CT are able to fully differentiate into skin equivalents in a 3D culture model when co-cultured with primary fibroblasts, primary MSCs, BJ-5ta, or hTERT-MSCs. To confirm the functionality of the co-culture models, both the primary keratinocytes and the Ker-CT air-liquid interface (ALI) co-cultures were subjected to a scratch assay. Re-epithelialization occurred in both cell lines, and interleukin 8 (IL-8) showed an increase in expression from day 0 to day 1 and 3, corresponding to migration of cells into the wound. The continuous nature of the Ker-CT cell line makes it an invaluable model for the research of keratinocyte biology, as it eliminates the issue of short life span and donor variation seen with primary cells.

Development and Characterization of an in vitro Co-culture Angiogenesis Assay System Using hTERT Immortalized Cells for High Throughput Drug Screening

Abstract: In this study, we established an in vitro co-culture model system using the aortic endothelial cell line TeloHAEC (hTERT immortalized human aortic endothelial cell line, ATCC® No. CRL-4052™) and the hTERT immortalized adipose-derived mesenchymal stem cell line hTERT-MSC (ATCC® No. SCRC-4000™). Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts; TeloHAECs co-cultured with BJ primary fibroblasts (ATCC® No. CRL-2522™) for 14 days in optimized ATCC angiogenesis medium formed fine tubular structures as shown by staining with CD31 endothelial cell marker. The tubule length elongated with increasing doses of vascular endothelial growth factor (VEGF), and tubule formation can be completely blocked by suramin in a concentration-dependent manner. Next, we introduced GFP into the TeloHAEC cell line (TeloHAEC-GFP, ATCC® No. CRL-4054™), allowing for real time visualization of angiogenesis when co-cultured with BJ fibroblasts; another hTERT immortalized cell line, hTERT-MSC, replaced the BJ-primary fibroblast in the co-culturing system. It was observed that the new model forms the tubular structures in less than 7 days, and also responds effectively to VEGF and compounds such as suramin. Further, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by positive αSMA staining (a marker of smooth muscle cells), this indicates that the system has physiological relevance. Therefore, the co-culture models developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying vascular biology, drug screening and tissue engineering.

Using LUHMES Cells as a Model System to Study Dopaminergic Neuron Cell Biology and Parkinson’s Disease

Abstract: Dopaminergic neurons play significant roles in motor, reward, and motivational behavior related circuits throughout the brain. To date, there are few continuous in vitro models available to laboratories in research, industry, and academia for studies related to basic dopaminergic cell biology or high throughput screening. Here, we propose the use of a human model system, LUHMES cells (ATCC® CRL-2927™), to study dopaminergic neuron cell biology and Parkinson’s disease. LUHMES cells are neuronal precursors derived from fetal ventral mesencephalon. Neuronal differentiation is governed by the termination of v-myc expression using low levels of tetracycline. During our characterization, we found that tetracycline induced differentiation resulted in consistent neurite outgrowth in LUHMES cells within two to four hours. One day post differentiation, cells displayed similar morphology, with several long processes protruding from the cell soma. Growth cones were often observed in early differentiated cultures. Immunocytochemistry in early differentiated cultures (Days in vitro, DIV 2-3) revealed low level expression of tyrosine hydroxylase; however, these levels were increased significantly by 7 DIV with many neurons expressing tyrosine hydroxylase. We also investigated dopamine transporter expression. Differentiated LUHMES cultures were positive for neuronal markers such as bIII tubulin and devoid of expression of traditional glial markers including GFAP and IBA-1. Both undifferentiated and differentiated LUHMES cells were easily transfected using basic eGFP constructs, although greater efficiencies were observed with the use of viral constructs. In summary, LUHMES cells are a suitable and robust in vitro model system for studying dopaminergic neuron cell biology and mechanisms underlying Parkinson’s disease.

Cell Line Genomic DNAs for the Molecular Diagnosis of Cancer

Abstract: Large-scale cancer genome programs have generated a rich data set comprising genetic abnormalities observed in thousands of clinical patient tumors, which provides a major opportunity for the molecular detection of cancer. However, the lack of controls for molecular tests has been a challenge. Because of the reproducible nature of the cell lines, genomic DNAs of fully characterized and authenticated cell lines provide a solution.

Genomic DNAs were extracted from over 70 commonly used human cancer cell lines derived from the breast, lung, colon, and pancreas, as well as hematopoietic and lymphoid tissue. Cancer gene mutations were identified by next-generation sequencing. Gene copy number changes were analyzed using the qBiomarker Copy Number PCR Assays kit (QIAGEN). Moreover, the selected cell lines were analyzed by quantitative polymerase chain reaction (qPCR), Western blot, and immunofluorescence (IF) staining to verify gene and protein expression mutation.

Abstract: Positive controls are essential for establishing assay performance and equipment efficacy. Yet, some food testing laboratories refrain from using bacterial strains as positive controls for fear of cross-contaminating their samples. Under the Food and Drug Administration (FDA) Food Safety Modernization Act, laboratories face an increasing number of regulations to expand testing for objectionable organisms. Control strains with unique, easily detectable traits which distinguish positive control strains from actual food contaminants can help differentiate true contamination from control strain cross-contamination.

In this study, we introduced shuttle vectors encoding either green fluorescent protein (GFP, Life Technologies) or NanoLuc® (Promega) into Escherichia coli strains, including Shiga toxin-producing O157 (stx1+, stx2+, eaeA+) and non-Shiga toxin-producing O157 (stx1-, stx2-, eaeA-), for use in food pathogen detection assays. Both reporters can be easily visualized without specialized detection equipment; GFP fluoresces when excited by UV light, while bacteria engineered with NanoLuc emit a strong light signal in the presence of a chemical substrate. Upon establishing the detectability of NanoLuc in the E. coli O157 strains, the reporter was transformed into the “Big Six” non-O157 E. coli strains (serogroups: O26, O45, O103, O111, O121, and O145) for use as reporter-labeled positive controls.

Novel Fluorescent Reporters for Studying Pathogen-host Interactions

Abstract: Fluorescent proteins, such as green fluorescent protein (GFP), have diverse applications in the basic and applied sciences. While GFP has been frequently used in eukaryotic systems, its applications have been limited in microorganisms due to a lack of broad-range molecular tools. In this study, we have developed a vector to express GFP in pathogenic bacteria for use in bacterial pathogenesis and pathogen-host studies. A shuttle vector encoding the GFP variant mut31 (pUCP18-MCSgfpmut3) was generated and successfully transformed into various Gram-negative opportunistic pathogens from the ATCC collection, including: Escherichia coli (ATCC® 25922™), Salmonella enterica (ATCC® 14028™), Shigella flexneri (ATCC® 12022™), Pseudomonas aeruginosa (ATCC® 10145™), and the P. aeruginosa type strain PAO1 (ATCC® 15692™). P. aeruginosa was used as a model to test the characteristics of the vector and sensitivity of detection using a fluorescence plate reader, microscopy, flow cytometry, and in vivo imaging systems.

Development and Verification of Synthetic RNA Controls for Determination of Influenza Virus Load

Abstract: Influenza is one of the most significant causes of acute respiratory infection worldwide. Rapid diagnostic tests for highly contagious pathogens, such as Influenza, are essential for decreasing the public health impact of emerging infectious diseases and bioterrorism agents. However, these tests require positive controls that are not always readily available. Consequently, if worldwide public health laboratories are unable to meet the costly regulations required for the import, transfer, and safe use of pathogens used as controls, then critical diagnostic, surveillance, and epidemiological information could be missed.

The use of in vitro synthesized viral RNA as a control would provide essential equivalency standards that would be accessible to any laboratory performing quantitative RT-PCR tests. Synthetic RNA controls are particularly useful for laboratories which lack appropriate biosafety containment facilities for propagating a particular pathogenic virus, or have difficulty gaining access to the organism in question due to international tightening of both import and export controls.

Development of Synthetic Molecular Standards for Dengue Virus

Abstract: Dengue fever is an acute illness caused by any one of four serotypes (1-4) of genetically related dengue viruses (DENV), with an estimated 390 million cases reported annually. Currently, quantitative RT-PCR (qRT-PCR) is the preferred method for the detection and quantification of DENV in clinical diagnostics and epidemiological surveillance. The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known viral genome concentration.

Native DENV RNA can be used as a standard for these assays; however, the full-length dengue viral RNA is on the Commerce Control List and requires a permit from the US Department of Commerce for international shipment. To make DENV RNA standards more accessible, ATCC has developed four synthetic molecular standards that represent DENV serotypes 1-4. Each standard contains short fragments from the capsid, membrane, and envelope genes of the DENV genome, as well as target regions encompassing the primer sequences from numerous published RT-PCR assays, including the DENV-1-4 Real-Time RT-PCR Assay developed by the CDC1. The synthetic RNA standards were quantified by Droplet Digital™ PCR (ddPCR™) in order to package precise copies of RNA. Moreover, given the inherent labile nature of RNA, a stabilization matrix was added to the quantitated RNA preparation. As compared to native RNA, these synthetic standards are easier to use as controls for qRT-PCR assays, exhibit less variability, have a longer shelf life, eliminate the need to culture viruses and can be used under BSL-1 conditions. Further, this synthetic quantitative RNA approach can be extended to other pathogenic viruses which are unculturable or need to be grown in a high-containment facility.

In the following proof-of-concept study, we amplified the synthetic molecular standards with the published primers from the CDC assay1 and Waggoner et al2, and used the generated standard curves to quantify viral RNA extracted from various DENV strains.

Abstract: Patient-specific induced pluripotent stem cells (iPSCs) provide a unique tool for the study of human diseases such as Parkinson’s disease. To provide a better research tool for studying Parkinson’s disease, we generated three iPSC lines, from dermal fibroblasts of a 63 year old Caucasian male, diagnosed with Parkinson’s disease, by reprogramming with sendai viral, retroviral, or episomal expression of OCT3/4, SOX2, KLF4, and MYC genes. The Parkinson iPSC lines generated with different reprogramming methods all demonstrated similar cell morphology, pluripotent marker expression, and the ability to differentiate into three germ layers. Compared to an hiPSC line-derived from a healthy subject, these Parkinson’s iPSC lines showed similar efficiency of neural differentiation into neural progenitors from iPSC-derived embryoid bodies. To more effectively model Parkinson disease, we have sequenced all exons of the three Parkinson iPSC lines along with their parent fibroblast by exome sequencing with an Agilent’s SureSelect 51 Mb array. Compared to the hg19 human genome reference, each cell line has over 300 genes with missense mutations and there are 226 genes with missense mutations conserved among all four cell types. More importantly, there are three amino acid changes within the LRRK2 gene, the most common Parkinson’s disease-related gene, at positions 50 (R50H), 723 (I723V), and 2397 (M2397T), which have previously been reported in Parkinson’s patients. Via integrating and non-integrating reprogramming methods, we have created three fully characterized iPSC lines that carry LRRK2 mutations.

Tumor Cell Panels: New Tools in Genomic Era

Abstract: Increased understanding of cancer genome is affecting every corner of cancer research. Although human tumor cell lines have been used as essential tools for decades, there are only a few cell line panels have been developed for the drug screening. There is a gap between the new knowledge of cancer genome and the cell line based platforms for both basic and translational research. Here, we show that new generation tumor cell panels are filling the gap. The panels were generated by selecting authenticated cell lines derived from variant cancer types, and annotated with genetic alteration information generated by large scale sequencing projects such as the Catalog of Somatic Mutations in Cancer (COSMIC) and the Cancer Cell Line Encyclopedia (CCLE). To capture the genetic diversity of cancer, each panel includes cell lines with varying gene mutation complexity. To further facilitate targeted drug discovery, the molecular signature tumor cell line panels focus on individual driver genes, critical protein kinases, transcription factors and cell signaling pathways. Those panels have been analyzed to verify gene mutation, gene expression, protein expression and bio-functions.

ATCC Vendor ShowcasePresentation at ASCB 2013

hTERT Immortalized Cells — Unique tools for tissue-relevant research

ATCC Human telomerase (hTERT) immortalized cell lines combine the in vivo nature of primary cells and the long culture life of continuous cell lines. This section will provide an overview of the hTERT-immortalized cell line collection, and will examine the use of RPTEC/TERT1 (ATCC® No. CRL-4031™) and TIME (ATCC® No. CRL-4025™) cell lines to demonstrate how hTERT-immortalized cell lines can help you reach your research goals.

ATCC has a wide selection of induced pluripotent and mesenchymal stem cells along with an array of associated culture media and reagents. This section will provide an overview of the ATCC stem cell collection and describe how these resources can be used in cell biology studies.

Transfex — for hard to transfect cells

ATCC offers a superior lipid-based transfection reagent (TransfeX) that can be used to transfect difficult cell types, like primary and stem cells, and uncomplicated continuous cell lines. In this section, we will show how TransfeX provides higher transfection efficiency and lower cytotoxicity than other commercially available transfection reagents. We will also describe the HEKPlus Expression System for protein expression.

ATCC Molecular Signature Panels — Powerful tools for the genomics age

ATCC molecular signature panels focus on key components of cell signaling pathways such as EGFR, AKT, PI3K, PTEN, or p53. This section will describe how we generated these panels using authenticated cell lines containing critical gene copy number changes and site mutations, as well as how each panel was experimentally validated for genetic alterations, protein expression, and cell functionality.